1. Field of the Invention
The invention relates generally to fluorometers, instruments for measuring wavelength and intensity of fluorescence. More particularly, the invention relates to a multiple protocol fluorometer (MPF) for measuring photosynthetic parameters of phytoplankton and higher plants using an actively stimulated fluorescence signal.
2. Description of the Related Art
Measurement of photosynthetic activity that occurs in photosynthetic organisms such as phytoplankton or higher plants is important in understanding the basic physiology of phytoplankton and higher plants, as well as to ecological studies of environmental stress. In ocean studies, for instance, characterization of photosynthesis of phytoplankton is useful in (i) understanding the ocean carbon cycle, (ii) predicting how climate-induced changes in ocean circulation, as well as, anthropogenic perturbation affect ocean productivity, and vice versa, and (iii) understanding how oceans can mediate climate change.
Furthermore, in terrestrial applications, measurements of photosynthetic parameters can be used to (i) assess effects of the environmental factors (nutrients limitation, drought, temperature) on plants' physiological performance, (ii) select and evaluate crop strains with respect to resistance to natural and anthropogenic stresses, (iii) assess physiological response of plants to contamination insults (chemicals, heavy metals, low level radioactive wastes), and (iv) evaluate the efficacy of remediation efforts with respect to restoring the ecological status of contamination threatened ecosystems.
Assessment of photosynthesis by photosynthetic organisms requires either a direct measurement or an indirect approach based on measurement of photosynthetic parameters. Direct measurements of photosynthesis of phytoplankton or higher plants include those of CO.sub.2 exchange, O.sub.2 evolution, or radioactive labelled carbon incorporation (i.e., .sup.14 C method). However, these measurements are laborious, time consuming, and not applicable in certain conditions. In studying phytoplankton, for example, the .sup.14 C measurement method requires an incubation and can be done only for discrete, bottled samples. Further, accuracy in photosynthesis measurements of phytoplankton in laboratory settings are limited as a result of removal of the phytoplankton from its normal ambient nutrient flux, and laboratory simulation of ambient light and temperature conditions. In terrestrial applications, where the CO.sub.2 exchange method is the only applicable measurement, extensive sample manipulations are required, resulting in a measurement process that itself modifies the investigated photosynthetic parameters.
Indirect measurements of photosynthesis, based on a functional relationship between photosynthetic activity and fluorescence, have proven to be more successful. Such indirect measurement methods include both passive fluorescence and active fluorescence techniques.
Passive fluorescence techniques are flawed by an assumption that the ratio of the photosynthetic to fluorescence yield is constant. In nature, this ratio can vary by as much as 10:1, making the passive fluorescence based estimates of photosynthesis unreliable. More detailed measurement and study of photosynthetic processes, such as light absorption, primary photochemistry, and electron transport between so-called Photosystem II (PSII), and Photosystem I (PSI), are not possible with passive fluorescence techniques.
Active fluorescence techniques, on the other hand, are based on flash stimulated fluorescence. An example employing an active fluorescence technique, is disclosed in Moll, U.S. Pat. No. 4,650,336, which describes a method and device for measuring photosynthesis, specifically variable fluorescence of plants. Variable fluorescence is measured as the difference between a low level, steady state fluorescence and a higher level of a fluorescent transient. The fluorometer device of Moll has one lamp to provide constant-level light to bring about continuous, steady state fluorescence of a plant, and a flash lamp to provide a flash of light (excitation energy) to bring about a transient fluorescence of the plant. The device and method of Moll utilize the second flash lamp to produce either a single flash, or series of flashes at a slow repetition rate, approximately one hundred (100) Hz. At 100 Hz, however, the flash rate is too slow to effectively measure the photosynthetic processes occurring in photosynthetic organisms.
Another active fluorescence technique is described in Kolber et al., U.S. Pat. No. 4,942,303 (Kolber I), the contents of which are incorporated, in its entirety, by reference herein. The technique described in Kolber I enables a more detailed measurement of photosynthesis. Specifically, the technique involves use of "pump and probe" flashes for measuring the change in fluorescence of phytoplankton or higher plants. A relatively low intensity probe flash is followed quickly by a pump flash that is usually made intense enough to saturate PSII.
Kolber I also discloses a computer controlled fluorometer device and method that measures photosynthesis by monitoring and recording changes in fluorescence produced by a computer controlled series of cycles of probe and pump flashes. From these measurements, various photosynthetic parameters can be determined and incorporated into a mechanistic model of photochemistry based on the kinetics of electron flow between PSII and PSI.
The pump and probe technique, although very successful in measuring the photosynthesis occurring in phytoplankton or higher plants, has the following operational limitations:
1. In order to measure the absorption cross-section and the rate of electron flow from PSII to PSI the pump and probe fluorometer employs a sequence of probe, pump, and probe flashes, repeated up to 30 times, with the intensity of the pump flash changed from zero to a supersaturating level, or with the delay between the pump, and the second probe flash changing from 80 .mu.s to 300 ms. These two protocols require 5 minutes to 10 minutes of fluorometer operation in order to make appropriate measurements. Particularly, when the pump and probe technique is used in a profiling mode for studying phytoplankton in the ocean, where these protocols often have to be executed at every meter of a water column, the time required for making the measurements is prohibitively long.
2. The intensity of the probe flash has to be kept below 1% of the PSII saturation level. This low intensity flash results in a low signal to noise ratio, particularly at low chlorophyll concentrations.
3. The pump and probe fluorometer requires two separate excitation channels (i.e., two flashers) which complicates construction, and increases the cost of the fluorometer.
4. Execution of a full experimental protocol, particularly in studying phytoplankton in the ocean, utilizes a large amount of electrical power. This requirement limits long-term, remote mooring applications where electrical batteries are used to power the fluorometer.
Kolber et al., U.S. Pat. No. 5,426,306 (Kolber II), the contents of which are incorporated, in its entirety, by reference herein, discloses a fast repetition rate (FRR) fluorometer that is operable to produce a series of fast repetition rate flashes in the range of 10,000 Hz to 250,000 Hz, and at controlled energies sufficient to gradually and incrementally effect photosynthetic processes occurring in PSII and PSI in phytoplankton or higher plants, for measurement of fluorescence with higher signal to noise ratios relative to the device of Kolber I. The FRR fluorometer of Kolber II, however, has the following disadvantages:
1. The methodology and the instrument disclosed in Kolber II has a limited resolution of the saturation and fluorescence decay, because the flash rate of the xenon flashlamp in the FRR fluorometer is limited to 250 kHz. When there is pre-triggering prior to each flash, the maximum flash rate falls below 10 kHz. Therefore, the FRR fluorometer is incapable of resolving the initial portion of the fluorescence transient that is critical for assessment of the faster photosynthetic processes such as the extent of energy transfer between PSII reaction centers. Due to the same limitation, only the early stage of fluorescence decay can be measured, thus limiting the resolution of the assessed kinetics of electron transport in PSII to a single time constant.
2. The power dissipation by the xenon flashlamp in the FRR fluorometer is excessive and limits the measurement protocols that can be used with the FRR fluorometer. For example, when generating flashes at intervals of 60-100 .mu.s, the xenon plasma must be reheated using about 2 .mu.s-long high current pulse (average 200 A at 400 V), dissipating about 0.16 J of energy per flash. The power rating of xenon flashlamp used in FRR fluorometer is about 15 W, thus limiting the number of flashes to less than 100 flashes. Furthermore, the excessive power dissipation limits long-term, remote mooring applications where electrical batteries are used to power the fluorometer, such as, for example, in a submersible instrument with a dual, self-cleaning chamber, discussed further herein.
3. Assessment of the correct functional absorption cross section, and the correct kinetics of electron transport within PSII and from PSII to PSI requires knowledge of the extent of energy transfer between PSII reaction centers, which cannot be measured with the FRR fluorometer.
4. The FRR fluorometer is incapable of spectrally resolving the functional absorption cross section or the extent of energy transfer between PSII reaction centers.
5. The xenon flashlamp in the FRR fluorometer cannot provide ambient illumination.
6. The xenon flashlamp in the FRR fluorometer is a pulsed light source using high voltage electronics in which large switching currents at high voltages cause a high level of RF noise.